Project Summary: Tick borne diseases continue to emerge in the United States. Powassan virus (POWV), transmitted by the Lyme disease vector Ixodes scapularis, is a member of the tick-borne encephalitis virus (TBEV) complex. It causes encephalitis, including fatal neuroinvasive disease in approximately 10% of cases, and long-lasting neurological sequelae have been documented in 50% of survivors. POWV has recently re-emerged, as evident by the increase in number and expanding geographic range of confirmed cases (USGS, 2015; CDC). POWV transmitted to humans by infected ticks, and successful transmission of POWV can occur within three hours of Ixodes scapularis attachment. Successful tick feeding is facilitated by a collection of pharmacologically active factors/proteins in tick saliva, which are secreted into the feeding pool that the tick creates on the vertebrate host. The skin serves as the interface of host-virus-tick interactions; therefore, the objective of this proposal is to investigate cutaneous host immunomodulation by POWV-infected ticks while examining components of tick saliva that lead to a more favorable environment for virus transmission, and identify salivary components that enhances POWV infection. We will test our hypothesis by exploring the following specific aims: (1) Investigate the role of POWV infected tick saliva in modulating immune responses at the bite site to facilitate POWV transmission and dissemination; (2) Identify the specific tick salivary protein/s that potentiate POWV transmission. The proposed research is innovative and novel because we, for the first time, will examine the molecular mechanisms involved in potentiation of a tick borne virus at the tick-host interface. We will also investigate concurrently both host and vector attributes of POWV infection using a systems biology approach. This has never been attempted before. The proposed project will also identify POWV infected cells at the site of tick feeding using Laser Scanning Cytometer that will provide us with precise identification of the type, number, time, and location of immune cells associated with early tick feeding time points. We will also identify the salivary protein/s that enhances POWV infection and dissemination, and also check if antibodies to these proteins will attenuate virus infection. This proposed work will make a significant contribution to the field because it will employ systems biology approach, cutting-edge technologies and innovative ideas to understand the mechanisms and identify salivary components that lead to a more favorable environment for tick-borne virus transmission. Overall, these studies will fill the gaps in our current knowledge/understanding of tick-borne viral infection that continues to increase in prevalence, and provide a new line of investigation of the tick-pathogen-host interface. This, in turn, will lead to development of countermeasures that will attenuate the virus infection.